Departments of Chemistry, Molecular Biosciences, and the Feinberg School of Medicine, Northwestern University, 2170 Campus Drive, Evanston, IL, 60208, USA.
J Ind Microbiol Biotechnol. 2014 Feb;41(2):451-9. doi: 10.1007/s10295-013-1373-4. Epub 2013 Nov 19.
The use of proteomics for direct detection of expressed pathways producing natural products has yielded many new compounds, even when used in a screening mode without a bacterial genome sequence available. Here we quantify the advantages of having draft DNA-sequence available for strain-specific proteomics using the latest in ultrahigh-resolution mass spectrometry for both proteins and the small molecules they generate. Using the draft sequence of Streptomyces lilacinus NRRL B-1968, we show a >tenfold increase in the number of peptide identifications vs. using publicly available databases. Detected in this strain were six expressed gene clusters with varying homology to those known. To date, we have identified three of these clusters as encoding for the production of griseobactin (known), rakicidin D (an orphan NRPS/PKS hybrid cluster), and a putative thr and DHB-containing siderophore produced by a new non-ribosomal peptide sythetase gene cluster. The remaining three clusters show lower homology to those known, and likely encode enzymes for production of novel compounds. Using an interpreted strain-specific DNA sequence enables deep proteomics for the detection of multiple pathways and their encoded natural products in a single cultured bacterium.
蛋白质组学用于直接检测表达天然产物的途径，即使在没有细菌基因组序列可用的筛选模式下，也产生了许多新的化合物。在这里，我们使用最新的超高分辨率质谱技术，对具有草案 DNA 序列的菌株特异性蛋白质组学进行了定量，该技术既可以用于蛋白质，也可以用于它们产生的小分子。使用链霉菌 NRRL B-1968 的草案序列，我们显示与使用公共数据库相比，肽鉴定数量增加了 >10 倍。在该菌株中检测到六个表达基因簇，它们与已知的基因簇具有不同的同源性。迄今为止，我们已经鉴定出其中三个簇编码生产灰绿霉素（已知）、雷卡菌素 D（一个孤儿 NRPS/PKS 杂合簇）和一种由新的非核糖体肽合酶基因簇产生的假定的 Thr 和 DHB 含有物。其余三个簇与已知的簇显示出较低的同源性，可能编码用于生产新型化合物的酶。使用解释的菌株特异性 DNA 序列可以在单个培养细菌中进行深入的蛋白质组学研究，以检测多种途径及其编码的天然产物。
